The focus of this project is to determine how mitochondria are optimized within muscle cells to help maintain energy homeostasis during the large change in energy demand caused by muscle contraction. Despite much interest, control of mitochondrial function in vivo remains largely unclear as direct measures of mitochondrial enzymes in live animals have been limited. We are currently developing multi-photon microscopy methods for evaluating mitochondrial function in the skeletal muscle of live mice to determine whether mitochondria within muscle fibers respond uniformly to muscle contraction as well as which signaling molecules are involved in regulating these processes. Additionally, mitochondrial function in live animals is intrinsically related to the content, location, and shapes of mitochondria within the cells. Thus, we are investigating how the grid-like mitochondrial networks observed in adult muscle cells are formed using three dimensional electron microscopy techniques together with multi-photon and super-resolution light microscopy. At the same time, we are developing transgenic mouse models to determine the physiologic function of proteins potentially involved in the rapid distribution of energy observed through the grid-like mitochondrial networks in skeletal muscle cells. Finally, we are using the genetically tractable Drosophila to determine how mitochondrial network shape and composition is determined in relation to the contractile apparatus during muscle development. These studies will provide insight into the mechanisms regulating energy distribution in skeletal muscle and help identify potential targets for therapeutic interventions related to obesity, aging, and other metabolic diseases.